Journal
METHODS
Volume 44, Issue 2, Pages 129-138Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2007.10.006
Keywords
acid urea polyacrylamide gel electrophoresis; Northern hybridization; aminoacylation; deacylation kinetics
Funding
- NIGMS NIH HHS [R37 GM017151, R01 GM067741-04, GM67741, R01 GM067741, F32 GM017151, R01 GM017151, GM17151, R56 GM017151, R37 GM017151-37] Funding Source: Medline
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Here we describe the many applications of acid urea polyacrylamide gel electrophoresis (acid urea PAGE) followed by Northern blot analysis to studies of tRNAs and aminoacyl-tRNA synthetases. Acid urea PAGE allows the electrophoretic separation of different forms of a tRNA, discriminated by changes in bulk, charge, and/or conformation that are brought about by aminoacylation, formylation, or modification of a tRNA. Among the examples described are (i) analysis of the effect of mutations in the Escherichia coli initiator tRNA on its aminoacylation and formylation; (ii) evidence of orthogonality of suppressor tRNAs in mammalian cells and yeast; (iii) analysis of aminoacylation specificity of an archaeal prolyl-tRNA synthetase that can aminoacylate archaeal tRNA(Pro) with cysteine, but does not aminoacylate archaeal tRNAcy' with cysteine; (iv) identification and characterization of the AUA-decoding minor tRNA(Ile) in archaea; and (v) evidence that the archaeal minor tRNA(Ile) contains a modified base in the wobble position different from lysidine found in the corresponding eubacterial tRNA. (C) 2007 Elsevier Inc. All rights reserved.
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